Field of the Invention
Embodiments of the present invention relate in general to the use of molecular inversion probe technology in capture sequence methods and amplification methods.
Description of Related Art
Molecular Inversion Probe (“MIP”) technology is a high-throughput genotyping technology capable of interrogating single nucleotide polymorphisms on a large scale. Methods of using molecular inversion probe technology in highly multiplexed genotyping of SNPs are known. See Hardenbol et al. Genome Res. (2005) 15:269 and Hardenbol et al. (2003) Nat. Biotechnol. 21:673. The use of molecular inversion probe technology in allele quantification is also known. See Wang et al. (2005) Nucl. Acids Res. 33(21).
Generally, MIP technology is directed to the use of a single oligonucleotide probe with recognition sequences at each terminus. The probe also includes a specific tag sequence that is ultimately read on a microarray, and two PCR primers that face away from each other and therefore cannot facilitate amplification. The probe is hybridized with a genomic target sequence such that it forms a circular structure, with the ends of the probe abutting. This leaves a single base gap at the location of a SNP. This gapped-duplex is then tested in four separate reactions, each with a single dNTP species present, in which successful polymerization and ligation provides allelic differentiation. The probes are subsequently released from the genomic DNA and those that have been covalently circularized in the correct allele/nucleotide reaction combination are amplified using a “universal” PCR primer pair. Each amplified probe contains a unique tag array referred to here as “barcoding.” Tags are selected to have a similar Tm and base composition and to be maximally orthogonal in sequence complementarity. Amplicons are fluorescently labeled and the tag sequences released from the genome homology regions using a restriction endonuclease treatment. The tags are then detected using a complementary tag array.